Part:BBa_K3017067
Construct for testing CRISPRi sgRNA cross-talk with non-target DNA
This asRNA characterization construct is to characterize the cross-talking by sgRNA mismatch with different FP.
asRNA is a purely synthetic RNA that is designed to bind with its paired sgRNA at its extensor region. By removing the secondary structure of sgRNA extensor, dCas9 leave the sgRNA. The CRISPRi repression is reversed by the asRNA.[1]
The asRNA characterization constructs all contain a constitutively expressed dCas9, a fluorescent protein RFP, asRNA under pBAD promoter, and sgRNA (GFP). Under the regulation of pBAD promoter, asRNA is transcripted when arabinose is added to the culture medium. The transcription start site of the pBAD promoter has been identified according to Brzozowska et al. (2018), in which the asRNA has been placed on the Transcription Start Site (TSS). For sgRNA transcription, weak Anderson promoter BBa_J23115 of strength 0.15 is used. Since the sgRNA transcribed will be targeting for gfp, suppression effect on the RFP should be non-desirable. This allows us to assess the cross-talking by sgRNA mismatch.
References:
[1] Y. J. Lee, A. Hoynes-Oconnor, M. C. Leong, and T. S. Moon, “Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system,” Nucleic Acids Research, vol. 44, no. 5, pp. 2462–2473, Feb. 2016.
[2] C. Anders, O. Niewoehner, A. Duerst, and M. Jinek, “Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease,” Nature, vol. 513, no. 7519, pp. 569–573, 2014.
[3] S. H. Sternberg, S. Redding, M. Jinek, E. C. Greene, and J. A. Doudna, “DNA Interrogation by the CRISPR RNA-Guided Endonuclease Cas9,” Biophysical Journal, vol. 106, no. 2, 2014.
[4] T. Karvelis, G. Gasiunas, A. Miksys, R. Barrangou, P. Horvath, and V. Siksnys, “crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus,” RNA Biology, vol. 10, no. 5, pp. 841–851, 2013.
[5] T. Møller, T. Franch, P. Højrup, D. R. Keene, H. P. Bächinger, R. G. Brennan, and P. Valentin-Hansen, “Hfq,” Molecular Cell, vol. 9, no. 1, pp. 23–30, 2002.
[6] G. M. Cech, A. Szalewska-Pałasz, K. Kubiak, A. Malabirade, W. Grange, V. Arluison, and G. Węgrzyn, “The Escherichia Coli Hfq Protein: An Unattended DNA-Transactions Regulator,” Frontiers in Molecular Biosciences, vol. 3, 2016.
[7]N. Brzozowska et al., “Characterizing Genetic Circuit Components in E. coli towards a Campylobacter jejuni Biosensor,” p. 290155, 2018.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 3336
Illegal PstI site found at 4758
Illegal PstI site found at 4962
Illegal PstI site found at 4992
Illegal PstI site found at 6204 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 919
Illegal NheI site found at 942
Illegal NheI site found at 2312
Illegal NheI site found at 2492
Illegal NheI site found at 2515
Illegal PstI site found at 3336
Illegal PstI site found at 4758
Illegal PstI site found at 4962
Illegal PstI site found at 4992
Illegal PstI site found at 6204 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2797
Illegal BamHI site found at 2251 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 3336
Illegal PstI site found at 4758
Illegal PstI site found at 4962
Illegal PstI site found at 4992
Illegal PstI site found at 6204 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 3336
Illegal PstI site found at 4758
Illegal PstI site found at 4962
Illegal PstI site found at 4992
Illegal PstI site found at 6204
Illegal NgoMIV site found at 3624
Illegal NgoMIV site found at 4728
Illegal NgoMIV site found at 4801
Illegal NgoMIV site found at 5286
Illegal NgoMIV site found at 6195
Illegal AgeI site found at 616
Illegal AgeI site found at 728
Illegal AgeI site found at 2086 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 2068
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